Transformation problems...

R. Jayakumar jakku at mrna.tn.nic.in
Sun Jan 16 09:47:30 EST 2000


hi
   It will be nice if I know what sort of transformtion you use.  Anyway, if
you are doing simple E. Coli transformation , then it could be just a
problem with your comp cells.  We follow a very simple protocol for our
transformation and it works every time.  Actually the simplicity may make
you a bit sceptical. But believe me, it works just great.

    ONE STEP TRANSFORMATION PROTOCOL FOR E.COLI

1. Inoculate E.coli (JM109 or DH5a etc..) in LB and subculture.
2. Reach the OD upto 0.3-0.4 (at 600nm) (for JM109.. it is around 2 hours)
(this is very important.
3. Harvest cells at 6K for 10min. at 4C.
4. Resuspend in 1/100th volume of TSS and freeze at -70C immediately.(We
store it as 100ul aliquots)  (eg. for 100 ml culture, suspend in 1ml of
TSS).
        YOUR COMP CELLS ARE READY.
5. Take a 100ul aliquot and add DNA (around 10-20ng) and leave on ice for
30min (we found that heat shock treatment gives less transformants than ice
treatment).
6. Add 900ul of LB or TSS (preferably) and gently invert.Incubate at 37c at
225rpm for 60 min. for expression
7. Plate the cells on appropriate media (we use LB).
    Efficiency is 1-4 X 10^6 cfu/ug of DNA.

TSS (Transformation and Storage solution) (for 100 ml).
Tryptone    - 1.0 gm
Yeast extract - 0.5 g
Nacl            - 1.0 g
PEG            - 10%
Mgcl2.6H2O - 1.0 g
DMSO        - 5%
D. water    - 90 ml.
pH 0 6.5

Note. Make up the solution, adjust the pH and then Add DMSO. Filter
sterilise TSS through a 0.2u filter.

    I have even made partial genomic libraries with this method.  It works
every time. I am sure you are going to get loaded with clones.
    as to your second part.. we rarely use deionised water for
transformation.  We only use sterilised double distilled water.  So no
problem there.   I think Millipore water is the best anyway. I use only
DDH2O.
  If anyone knows the author of this protocol, please let me know.  I got it
from a protocols book and I missed the name somewhere.
  Best of luck

sincerley
jayakumar
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R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at usaf.org
tel: +91-452-858471-374
efax: (603)-688-4665
web: http://members.tripod.com/~jakspage
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"There is a possible even in imPOSSIBLE"

----- Original Message -----
From: khalil <khalilha at sympatico.ca>
To: <methods at hgmp.mrc.ac.uk>
Sent: Sunday, January 16, 2000 9:46 AM
Subject: Transformation problems...


> Hi everyone !
>
> I am writing on behalf of an entire departement of microbiology which
> students are loosing all hopes. We are at least 7 students in 5 different
> laboratories of the university who are not able to have a single colonie
> after transformation.
> We would like to know if anyone ever encountered such a problem. Please
> avoid any joke about our ability to transform, we all have either master's
> or PhD. This week we decided to look at what we did in common in our
> experiments that would be the cause of our problems. The only thing that
we
> found would be the water we use (millipore milli-Q). We all get our
> deionised water from the same source in the departement. Could it be the
> cause, a problem with the filters or anything ? while we are trying to
> find-out, any help would be greatly appreciated. Did anyone ever had
> problems of transformation due to water. Thank you in advance.
>
>
>

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